Cell line specific effects of the interferons are mediated through the coordinated induction of a subset of genes expressed at precise times during the cytokine response. The principal investigator has shown that p15, a 17 kDa early gene product of type 1 interferon induction, bears marked homology to a tandem di-ubiquitin sequence. Subsequent evidence suggests the biological response of this Ubiquitin Cross Reactive Protein (UCRP) is mediated through covalent ligation to a small subpopulation of intracellular target proteins. Recent results indicate UCRP conjugation to target proteins proceeds through a ligation pathway distinct from that of ubiquitin. This novel mechanism for interferon action will be examined in six specific aims. (1) Physical characterization of UCRP-- the stability of recombinant UCRP and its precursor to various structural perturbants will be monitored by CD and fluorescence quench for comparison to ubiquitin. (2) Examine the binding of UCRP to intermediate filaments--transient expression of UCRP-chloramphenicol aminotransferase (CAT) in cultured A549 cells will be used to confirm earlier immunohistochemical data suggesting UCRP serves as a trans acting binding determinant for noncovalent association of target proteins with intermediate filaments. Deletion analysis of UCRP-CAT constructs will be utilized to identify the binding motif on UCRP responsible for filament association. Partial microsequencing will be used to characterize three novel low molecular weight (15-17 kDa) intermediate filament-associated proteins found to bind UCRP. (3) Examine the dynamics of intracellular UCRP pools--direct assays and complementation studies of (125)1-UCRP conjugation in cultured A549 cell extracts will examine the regulation of the ligation reaction during interferon-beta induction. Potential roles for UCRP in the interferon response will be tested by blocking UCRP synthesis through transient expression of antisense UCRP MRNA. (4) Purify and characterize the preUCRP processing activity. (5) Examine the enzymology of UCRP conjugation--in vitro (125)1-UCRP conjugation assays will be employed to identify the enzymes(s) present in A549 extracts catalyzing polypeptide ligation. These enzymes will be purified and characterized for comparison to the ubiquitin conjugation pathway. (6) Cloning of the UCRP conjugating enzymes--partial microsequencing of the UCRP conjugating enzymes will be used to construct probes for screening an interferon-induced A549 cDNA library. Cloning and expression of the UCRP conjugating enzymes for subsequent mechanistic studies will provide functional comparisons to the parallel ubiquitin ligation pathway.